Two-photon laser scanning fluorescence microscopy, first demonstrated by Denk et al. in 1990, is based on the simultaneous absorption of two low-energy photons by a fluorophore in a single quantum event to induce an electronic excitation that is normally accomplished by a single high-energy photon. The main advantage of two-photon microscopy over conventional linear microscopy stems from the high spatial localization of this excitation event due to the quadratic relation between the excitation and illumination intensity. Practically, this means fluorescence occurs only at the beam focus and as ultrafast, pulsed, near-IR laser irradiation is used as the excitation source, deeper imaging into optically thick tissue, with restricted photobleaching and phototoxicity to the exposed area of the specimen is achieved. The tenants of two-photon absorption (2PA) enables investigations of complex biological problems and experiments on living samples not possible with other imaging techniques, and highlighted examples of its use in the medicinal and biological research areas have been noted.
Two-photon fluorescence methods and techniques have advanced to yield sophisticated imaging techniques, but conventional UV-excitable fluorophores not optimized for efficient 2PA continue to be utilized. Two-photon photophysical characterization databases for commonly used fluorophores and bio-indicators have been reported. Many of these compounds exhibit relatively low two-photon absorption cross-sections (δ), on the order of 10 to 100 GM units. The (δ) parameter is an indicator of the two-photon absorption efficiency, and only recently has research been reported on the design and development of very efficient 2PA dyes on the order of >10 GM units. Much of the synthetic efforts have focused on preparing new organic dyes for a range of applications, as molecules specifically engineered for two-photon excitation may significantly outperform standard fluorophores optimized for single photon excitation. However, the identification and availability of optimized 2PA fluorophores specifically tailored for labeling biomolecules for two-photon fluorescence imaging studies are rare. To date, reactive fluorescent dyes exhibiting high 2PA cross-sections, specifically to covalently modify biomolecules have not been reported. Hence, the need to incorporate reactive, efficient 2PA fluorophores for covalent attachment onto biomolecules within the fluorophore design strategy seems timely and fulfills an appropriate need which coincides with increasing usage of two-photon excitation fluorescence imaging applications in the life sciences.
Previous studies have reported on the design and development of fluorene-based organic dyes with very efficient two-photon absorption and fluorescence emission properties and their use in data storage and sensing applications.
It would be highly desirable to provide a two-photon probe with the properties of high fluorescence quantum yields, of high photon absorptivity and high photostability.